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Short Communication |
Warwick HRI, University of Warwick, Wellesbourne, Warwick CV35 9EF, UK
Correspondence
Eugene V. Ryabov
eugene.ryabov{at}warwick.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession no. of the sequence reported in this paper is EF517277.
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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)] and four RNA viruses. The latter include aphid lethal paralysis virus (ALPV) (van Munster et al., 2002) and Rhopalosiphum padi virus (RhPV) (Moon et al., 1998), both members of the family Dicistroviridae, which infect a variety of insect hosts, and the unclassified Acyrthosiphon pisum virus (APV) (van der Wilk et al., 1997) together with the closely related rosy apple aphid virus (our unpublished data). These viruses were isolated from laboratory cultures of aphids from which significant quantities of virus could be obtained. However, the spectrum of viruses in laboratory cultures may not reflect that present in nature and also decreases the possibility of detecting viruses that cause pathology. In addition, it is difficult to use field-collected aphids for this purpose because field colonies often contain very few insects. To overcome this problem we have developed a novel approach to amplify viral nucleic acid from a small number of field-collected aphids, which has been used to identify the first aphid iflavirus, Brevicoryne brassicae virus (BrBV). Here we report the complete nucleotide sequence and describe the genome organization of BrBV and evidence that BrBV replicates in B. brassicae.
Amplification of the encapsidated RNA was adapted from the procedure described by Allander et al. (2005). Fifty adult B. brassicae aphids (total weight 15 mg), collected from different locations in Warwickshire, UK, during the summer of 2005 and stored at –80 °C, were homogenized with 15 ml 10 mM sodium phosphate buffer, pH 7.5. The homogenate was then centrifuged at 350 g for 10 min in a bench top Eppendorf centrifuge. The debris-free supernatant was filtered through a 0.80/0.22 µm filter (Millipore) and then centrifuged at 45 000 r.p.m. in a Ti80 rotor (Beckman) at 4 °C for 120 min. In order to remove traces of DNA contamination, 100 units of DNase I (Stratagene) were added and the sample was incubated at 37 °C for 30 min. RNA was extracted from 50 µl of the resuspended pellet with an RNeasy kit (Qiagen) according to the manufacturer's instructions and eluted with 50 µl RNase-free water. A 10 µl aliquot of the RNA was used for the reverse transcription and random amplification of encapsidated RNA using the method described by Allander et al. (2005), which involved cDNA synthesis using the tagged random hexanucleotide 5'-GCCGGAGCTCTGCAGATATCNNNNNN-3' for both the first- and second-strand cDNA synthesis and subsequent amplification of the cDNA with the primer 5'-GCCGGAGCTCTGCAGATATC-3'. The PCR products were separated in an agarose gel and the products, ranging in size from 150 to 400 nt, were isolated and cloned into the pDrive vector (Qiagen) to create a plasmid library. In total, 95 clones were sequenced and a sequence similarity search was carried out using the BLAST program (Altschul et al., 1990). Three clones (Fig. 1a) contained cDNA inserts, potentially encoding peptides showing sequence similarity with the polyproteins of members of the genus Iflavirus, sacbrood virus (SBV) (aa 248–325 and 2384–2457) (Ghosh et al., 1999), Varroa destructor virus 1 (VDV-1) (Ongus et al., 2004), Kakugo virus (KV) (Fujiyuki et al., 2004) and deformed wing virus (DWV) (Lanzi et al., 2006) (aa 314–392 and 2384–2491). The majority of the other clones contained insect or bacterial rRNA sequences.
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). Specific primers were used to amplify the 6.0 kb central region between the primarily identified BrBV cDNA clones (Fig. 1a
) and for the cloning of the 5'- and 3'-termini. The presence of the poly(A) sequence at the 3'-terminus of BrBV, a characteristic feature of iflavirus genomic RNA (Christian et al., 2005), allowed amplification of the 3'-terminal part of BrBV using a tagged oligo dT primer. The T4 RNA-ligase-mediated RNA circularization of the proteinase K-treated BrBV RNA followed by RT-PCR amplification (Ghedin et al., 2005) and the primer extension with reverse transcriptase were carried out to produce the draft sequence of the 5' part of BrBV RNA. The complete sequence of the virus genome was determined using three long overlapping cDNA clones, p3.0 kb, p6.0 kb, and p8.5 kb (Fig. 1a), which were produced by RT-PCR.
The genomic RNA of BrBV is 10 161 nt long, excluding the 3'-terminal poly(A) sequence. A single open reading frame (ORF), nt 793–9744, encodes a predicted polypeptide of 2983 aa (Fig. 1a), with a 791 nt 5' non-translated region (NTR) and a 417 nt 3' non-coding region, which precedes the poly(A) sequence. The BrBV RNA, like other iflaviruses, has a low G+C content (31 mol% A, 35 mol% U, 16 mol% C and 18 mol% G). The extended 5'-NTR of BrBV RNA is likely to function in RNA replication and as an internal ribosome entry signal (Ongus et al., 2006). The secondary RNA structure prediction for the BrBV 5' NTR carried out by Mfold algorithm (Zuker, 2003) showed the presence of a number of structural elements. A 5'-proximal hairpin structure formed by nt 7–38 contains a 14 nt stem with the 5'-AUUU-3' loop [free energy –24.5 kcal mol–1 (–102.5 kJ mol–1)] and resembles the hairpin structures found close to the 5' termini of the genomic RNAs of Perina nuda virus (PnV) and Ectropis obliqua picorna-like virus (EoPV) (Ongus et al., 2006; Wang et al., 2004; Wu et al., 2002). Apart from the 5'-terminal hairpin element, other predicted structural elements of the BrBV 5'-NTR include a nt 40–149 hairpin [free energy –36.0 kcal mol–1 (–150.7 kJ mol–1)] and two complex structures, nt 310–581 [free energy –71.2 kcal mol–1 (–298.0 kJ mol–1)] and nt 645–766 [free energy –28.5 kcal mol–1 (–119.3 kJ mol–1)], which do not have much in common with those identified in other iflaviruses (Ongus et al., 2006). High diversity of the 5'-NTR secondary structure elements has been reported, for example, in the family Picornaviridae (Witwer et al., 2001).
Sequence analysis of the predicted BrBV polyprotein showed that its N-terminal part has the highest similarity with the structural proteins of VDV-1, DWV and KV. The areas of highest similarity between BrBV polyprotein and the structural proteins of VDV-1 and DWV include the N-terminal peptides of the VP2 and VP1 proteins (Fig. 1b). It is therefore possible that proteolysis of the BrBV VP1 and VP2 homologues and the overall architecture of the BrBV virus particle are similar to those of VDV-1 and DWV. The C-terminal part of the BrBV polyprotein contains regions of sequence homology to the crucial catalytic amino acids of the RNA helicase (Fig. 1c), a chymotrypsin-like protease (Fig. 1d) and an RNA-dependent RNA polymerase (RdRp) (Fig. 1e) of picorna-like viruses (Koonin & Dolja, 1993), including iflaviruses, dicistroviruses and picornaviruses. The genome organization of BrBV is therefore typical for that of picorna-like viruses, including iflaviruses. The expression of the BrBV genes is likely to involve the expression of the single large 339 kDa polypeptide, followed by post-translational cleavage to produce the viral proteins (Christian et al., 2005). The BrBV polyprotein shows the highest level of similarity with the proteins of Hymenoptera iflaviruses: bee viruses DWV, VDV-1, KV and a virus of lepidopteran parasitoid wasp Venturia canescens picorna-like virus (VcPV) (Reineke & Asgari, 2005), (Fig. 2a). Indeed, the phylogenic analysis carried out using the RdRp domains of BrBV (aa 2633–2968) and other iflaviruses, dicistroviruses and picornaviruses showed that BrBV clustered (with a bootstrap value of 67 %) with DWV, VDV-1, KV and VcPV (Fig. 2b).
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) dissimilar to aphid rRNAs.
Aphids from a wasp-free laboratory culture were tested to find out whether BrBV is an aphid virus. In the culture of B. brassicae five out of five adult aphids tested contained similar levels of BrBV RNA, ranging from 11 to 43 pg per aphid (mean±SD is 22.8±12.1). No BrBV RNA was detected in aphids from the BrBV-negative B. brassicae culture reared at Warwick HRI for 3 years. No parasitoid wasp rRNA was detected in either the BrBV-positive or BrBV-negative cultures of aphids. Analysis of BrBV accumulation in a field wasp-infested population of B. brassicae was carried out to assess the incidence of BrBV in nature and the possible influence of BrBV on the aphid–wasp interaction. Of the 88 aphids tested, eight were BrBV-free and the rest showed a variety in BrBV RNA accumulation (from 0.001 to 28.0 pg per aphid). Eleven of the BrBV-positive aphids had levels similar to those found in the BrBV-positive laboratory culture (from 10 to 28 pg per aphid). Wide variations in virus accumulation were reported for another iflavirus, DWV (Yang & Cox-Foster, 2005; Yue & Genersch, 2005). Parasitoid wasp rRNA was detected in 23 out of 88 adult field-collected aphids. The sequencing of the wasp rRNA fragments (GenBank accession nos EF525172 and EF525173) revealed that the aphids were parasitized with a member of the Aphidiinae subfamily (Sanchis et al., 2000). Table 1 clearly shows that the aphids not infected with BrBV had the highest proportion of wasp rRNA-positive aphids. This was supported by the observation that the aphids with the highest accumulation of BrBV RNA had the lowest proportion of parasitoid-positive individuals.
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). Although certain endoparasitoid wasps have developed mechanisms to suppress the defence systems of their hosts, initiating virus replication (Renault et al., 2005), endoparasitoid infestation did not result in an increase in BrBV replication in B. brassicae. Moreover, the analysis of a natural population of B. brassicae indicates that adult aphids with higher levels of BrBV accumulation are less likely to be parasitized (Table 1). It is possible that the presence of BrBV has a negative effect on parasitoid wasp larvae development, or that parasitoid wasps avoid laying eggs into the aphids with high BrBV levels. Iflaviruses were shown to infect evolutionarily distant invertebrates, for example VDV-1 and DWV replicate in mites and honeybees (Ongus et al., 2004; Yue & Genersch, 2005) and VcPV replicates in both parasitoid wasp and lepidopteran host (Reineke & Asgari, 2005). BrBV does not appear to be lethal to aphids laboratory cultured for 7 months; however, in the field lethal effects cannot be excluded. In conclusion, BrBV is, to the best of our knowledge, the first iflavirus identified in aphids. Replication of this virus is associated with the aphid host and not the aphid host plants. The effects of the BrBV infection on B. brassicae and its parasitoid(s) require further investigation.
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ACKNOWLEDGEMENTS |
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Received 3 April 2007;
accepted 11 May 2007.
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